PROJECT SUMMARY Reduced serum levels of the inhibitor neurotransmitter gamma-aminobutyric acid (GABA) are found in patients suffering from progressive multiple sclerosis (MS), and preliminary evidence suggests that intestinal GABA levels are also reduced when compared to healthy individuals. Intestinal bacteria play a role in GABA and L-Glutamate, metabolism. GABA is an inhibitory neurotransmitter and key regulator of the gastrointestinal tract function and Glu is an excitatory neurotransmitter. Several groups of bacteria, including lactic acid bacteria synthesize Glu or harbor the enzyme glutamic acid decarboxylase (GAD) that catalyzes the synthesis of GABA. Because of their role modulating GABA/Glutamate levels, intestinal microbes can be targeted for GABAergic, inhibitory, effects. Our preliminary data show that disease, alone, alters the composition of the microbiota of experimental autoimmune encephalomyelitis (EAE) mice, a model to study MS. Among others, bacteria taxa grouped as lactic acid bacteria that contain species associated with immunomodulatory roles were reduced in the gut of diseased mice. We propose that the gut microbiota and its impact on systemic and neural GABA metabolism are key contributing factors to the clinical transition to the progression of MS. We hypothesize that, 1) the heightened immune/inflammatory response observed in patients who have experienced acute inflammatory MS increases intestinal barrier permeability; 2) this disrupts the composition of the microbiome to reduce the number of GABA-producing microbes; 3) Decreases in intestinal and systemic GABA concentrations follow, leading to, 4) enhanced immune/inflammatory response in the central nervous system (CNS) and the gut, resulting in additional further imbalances of both microbiota and GABA. In order to test our hypotheses, we propose to determine in the experimental EAE disease (C57BL/6 model induced with myelin oligodendrocyte glycoprotein 35-55, MOG35-55), whether GABA supplementation using a genetically engineered probiotic designed to express additional copies of GAD (GAD-L. lactis) will decrease inflammation by promoting immunomodulation (Aim 1), reduce intestinal barrier permeability (Aim 2) and ultimately improve disease progression (Aim 3). We anticipate that targeting GABA levels in EAE will reduce the extent of intestinal and systemic inflammation, intestinal permeability and bacterial translocation and will restore the composition of the intestinal microbiota. We predict that the outcome will be the protection against the progression of the disease, associated with enhanced levels of GABA both intestinal and systemically. Our research approach targeting the microbiota has not yet been attempted, underscoring the innovation and novelty of our project that could revolutionize therapeutics for MS. Our approach was designed specifically to offer undergraduate and graduate students the opportunity to contribute on a neuroimmunology project that focuses on the gut/brain axis.